Effects of Time-Restricted Eating on Nonalcoholic Fatty Liver Disease

Key Points Question Is time-restricted eating more effective in improving nonalcoholic fatty liver disease than daily calorie restriction? Findings In this randomized clinical trial including 88 patients with obesity and nonalcoholic fatty liver disease, the intrahepatic triglyceride content was reduced by 6.9% in the time-restricted eating group and 7.9% in the daily calorie restriction group during 12 months, but with no significant between-group differences. Time-restricted eating also did not produce additional benefits for reducing body fat or major metabolic risk factors compared with daily calorie restriction. Meaning The findings of this randomized clinical trial support the importance of caloric restriction with use of time-restricted eating among adults with obesity and nonalcoholic fatty liver disease.

117 DCR group at a significance level of 0.05 using a 2-tailed test. Thus, 34 subjects in each group are required. Assuming a dropout rate of 118 20%, we will recruit 43 subjects to each group in the proposed clinical trial.

128
• History of HIV, or active pulmonary tuberculosis;

129
• Diagnosis of type 1 and type 2 diabetes;

133
• Significant alcohol consumption in the past six months (Consumed more than 20 g/day for women or 30 g/day for men);

134
• History of serious cardiovascular or cerebrovascular disease (angina, myocardial infarction or stroke) in the past 6 months;

135
• History of severe gastrointestinal diseases or gastrointestinal surgery in the past 12 months;

137
• Being a smoker or having been a smoker in the 3 months prior to their screening visit;

138
• Taking medications affecting weight or energy intake/energy expenditure in the last 6 months, including weight loss medications,

139
antipsychotic drugs or other medications as determined by the study physician;

140
• Currently participating in weight loss programs or weight change in the past 3 months (> 5% current body weight);

141
• Women who are pregnant or plan to become pregnant;

142
• Patients who cannot be followed for 24 months (due to a health situation or migration);

143
• Patients who are unwilling or unable to give informed consent.

Randomization and masking
146 Randomization will be conducted by the Nanfang hospital of Southern Medical University in Guangzhou, China. The 147 randomization scheme is generated with SAS PROC PLAN (SAS Institution Inc). Although this is an open-label trial, the research 148 assistants who collect study outcome data will be masked to participants' intervention assignment.  All participants will be instructed to follow a diet of 1500-1800kcal/d for men and 1200-1500kcal/d for women (40-55% of energy 154 as carbohydrate, 15-20% as protein, 20-30% as fat) (28). Participants assigned to the TRE group will be instructed to eat from 8:00 155 AM to 4:00 PM and only noncaloric beverages is permitted outside of the eating window. Participants in the DCR group will be 156 instructed to follow a calorie restriction diet without restricting eating period. All participants will be provided with one protein shake 157 (Nutriease, Zhejiang Nutriease Co. Ltd., China) (

163
Participants will be encouraged to weigh foods to ensure accuracy of intake. All participants will be required to write their dietary 164 log and record daily food picture and mealtime on a custom mobile study application (App), which will be reviewed daily by study 165 nutritionist. Caloric intake assessment will be conducted by the nutritionist based on each participant's log and daily food photos based 166 on the nutrient content listed in the China Food Composition Table (30). Intervention participants will receive follow-up telephone calls 167 or App message twice per week, and meet with the dietician or nutritionist individually every 2 weeks to assess their adherence to the 168 program and provide suggestions for improvements and personalized energy targets (Table 2).

208
Whole body compositions will be quantified using a whole-body dual x-ray system (Lunar iDXA, GE Healthcare).

210
The bioelectrical impedance method will be used to measure body fat rate using the human body fat analyzer (V.Body HBF-371,

211
Omron). Visceral and subcutaneous fat area will be measured using abdominal CT (Revolution, GE Healthcare) at the level of the lumbar 214 vertebra(31). 215 9.8 The IHTG content:

217
Regions of interest (ROIs) will be selected in in the left liver lobe, the middle of the right liver lobe, and the posterior portion of the 218 right liver lobe in the transverse sections through the right hepatic portal vein and below the second hepatic portal vein(32-34). All the 219 selected ROIs should include hepatic parenchyma only, and exclude any biliary, vascular, or extrahepatic structures. The IHTG content 220 is calculated as the mean of values from five ROIs. The time points for baseline and outcome measurements are shown in Table 4.

232
Protection of participants from risks related to the intervention is of paramount concern to the physicians. All participants will be 233 evaluated for their medical history before enrollment. Contact information of the physicians are available for all the participants.

234
All participants should be contacted regularly to evaluate the occurrence, severity, and duration of physical discomfort, including 235 dizziness, fatigue, abdominal pain, headache, appetite change, constipation, or other adverse events during intervention. Evidence of the 236 occurrence of adverse events should be based on participants' self-report rather than suggestive questioning. Any medical adverse events 237 will be recorded and the nature, severity, and cause-effect relationship will be evaluated by physicians and recorded in a timely manner 238 on case report forms. The severity of adverse events is assessed as follows:

241
Mild: usually temporary, and not affecting daily activities;

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Moderate: causing discomfort and affecting normal activities, thought tolerable and not requiring participants to take any 243 medication.

244
Severe: disrupting daily activities and intolerable, requiring participants to take medication immediately.

245
With regard to the causal-effect relationship, medical adverse events are divided into five categories: definite, probable, possible, 246 unlikely, and unrelated. Only "definite" and "probable" are counted as adverse events. All case report forms for each participant should be filled out by study staff in a timely manner. The case report form should be 255 double-checked for potential errors or missing data prior to patients leaving the clinic. All data, including screening assessments, 256 questionnaires, physical examinations, and laboratory examinations, will be filed in the participant's chart. Original documents, 257 participants' charts, and CRF forms will be stored in the study office.

259
All data will be double-entered by researcher staff. Two sets of databases will be generated and tested for consistency using the 260 SAS program. Whenever inconsistencies are found, the data will be corrected by re-examination of the original case report forms or 261 laboratory reports.

263
Several standardized reports will be generated as follows: 1) participant recruitment and follow-up; 2) demographics; 3) data quality 264 and monitoring; 4) adverse events. These reports will be used for study management. These reports will be blinded to research personnel 265 who collect study outcomes.

268
Data will be analyzed according to participants' randomization assignments, regardless of their subsequent status (intention-to-

269
Original protocol Version 1.0 11 treat). PROC MIXED of SAS statistical software, version 9.4 (SAS Institute Inc), will be used to obtain point estimates and SEs of the 270 treatment effects and to test for differences between treatments. Group differences in the study outcomes will be evaluated using the 271 general linear model for continuous variables and the chi-squared test for categorical variables. A mixed-effects model will be used to 272 assess the effects of diet programs on the change in IHTG content and main outcomes and an autoregressive correlation matrix will be 273 used to correct within-participant correlation for repeated measurements. In this model, participants will be assumed to be random effects,

274
and intervention group, time, and their 2-factor interactions will be assumed to be estimable fixed effects. Multiple imputations for 275 missing data in the multivariable analyses will be conducted using the Markov chain Monte Carlo method. Data will be presented as 276 least-squares means with 95% confidence intervals (CIs). P < 0.05 will be considered statistically significant.

279
All study staff (physicians and nurses) will be trained and certified before the study is initiated. The training program will include 280 instruction on questionnaire administration and anthropometrics (height, weight, waist circumference, and hip girth) and blood pressure 281 measurement. A standard questionnaire will be used, which includes personal information (name, sex, age, place of birth, marital status, 282 occupation, education, household income, etc.), medical history, medication use, family history, lifestyle factors (smoking and drinking), 283 physical activity, dietary habits, and menstrual and reproductive history. Anthropometric measurements and blood pressure will be 284 obtained using a standard protocol. The data, including that for exercise intensity, duration, heart rate, and anthropometric measurements,

285
will be collected and recorded in CRFs by nurses every week. All technologists are experienced and masked to participant randomization.

288
China's Standard for Quality Management of Clinical Trials (GCP) and other relevant laws and regulations on clinical trials will 289 be complied with. The standard quality control process will be carried out at each step of the study, including repeated laboratory 290 measurements in 5-10% of random blind samples, and all data will be entered independently.

291
Establish a research procedure manual (MOP): we will develop a standardized process of data collection, which will include the 292 recruitment of subjects, instructions for the use of tables, intervention programs, index measurement, specimen collection and 293 management, etc., to guide all tabular information entry and diagnosis and treatment processes, and other aspects of the study.

294
Training: all researchers will receive relevant training before the start of the study, including standardized processes, recruitment 295 processes, follow-up, intervention programs, measurement procedures, and the use of MOP. At the same time, regular retraining sessions 296 will be conducted to ensure the quality of research.

297
Quality monitoring and reporting: quality control personnel will regularly review the QC data, including the timeliness of the 298 completion of the visit, data collection, program implementation and data quality.

301
A steering committee (Steering committee, SC), chaired by Drs Huijie Zhang and Yikai Xu, Nanfang Hospital, Southern Medical 302 University, will be responsible for the study. The research team consists of researchers and health management team members from the 303 Norte Health Management Center. the steering committee (SC) is responsible for organizing and supervising research implementation, 304 approving the research program, including modification of the research program, monitoring the recruitment and withdrawal process of 305 subjects, implementation of intervention programs, recruitment and intervention of subjects, data collection and quality control, and 306 responding to questions raised by the ethics body review committee (Institutional review board, IRB), etc. The research center will be 307 involved in the recruitment, intervention, data collection and quality control of the subjects. the research center will do randomization 308 and data analysis, and help prepare to report research results and papers for publication. In order to facilitate communication between 309 investigators and researchers, regular working meetings will be held. SC will discuss relevant scientific and management issues in the 310 study with three live meetings a year and monthly conference calls. Among them, important information, such as recruitment progress,

12
data completion and quality, and compliance with intervention programs, will be sent monthly by e-mail to committee members and key 312 staff.

315
This clinical trial must comply with the Helsinki Declaration (1996 edition) and the relevant regulations of Chinese clinical trials.

316
Before the trial begins, the hospital ethics committee of the research unit shall review the trial plan and issue the approval document 317 before the implementation of the trial plan.

318
For all research program amendments (excluding administrative amendments and amendments that have no impact on the 319 implementation of subjects, data or trials), the amendment and the applicable informed consent must be submitted to the Ethics 320 Committee for approval immediately prior to the implementation of these changes. The researcher is responsible for ensuring that the 321 conditions approved by the study are met and that the trial correction program or serious adverse events are reported to the ethics 322 committee or the corresponding organization as required by the ethics committee.

323
All subjects will be given the opportunity to read the entire informed consent document and, by the researcher or authorized 324 researcher, to provide the subjects, in writing, with a complete and comprehensive presentation of the background, purpose, research 325 methods, research process, test items to be involved, individual rights and obligations, possible risks, and possible benefits of the research 326 process. The subjects had enough time to ask questions, and the researchers needed to provide truthful and accurate answers in plain 327 language. The subjects had sufficient time to discuss with other family members whether to participate in the study. The subjects should 328 know that they can withdraw from the study at any time without any reason; the subjects agree to collect and apply the data related to 329 the study and are willing to cooperate with the follow-up. When they exit, the subjects are asked whether they agree to apply the collected 330 data. Participants will be required to sign informed consent before they can be selected for clinical trials. Informed consent shall be kept 331 for reference as one of the original materials for clinical trials.

334
The clinical study is expected to take about 3 years after registration in the clinicaltrials.gov (Identifier: NCT03786523), the trial 335 is expected to take about 3 years to carry out this clinical study, in which MOP development and personnel training and subject

456
can progress to steatohepatitis, cirrhosis or even liver cancer (3-5). Furthermore, NAFLD is considered a risk factor for type 2 diabetes 457 and cardiovascular disease (6).

458
Diet, sedentary lifestyle, and genetic predisposition have been associated with increased risk of NAFLD (6). With the 459 progressive global epidemic of obesity, it is expected with increased risk of NAFLD will rapidly increase. Previous studies indicated 460 that obesity was a major risk for NAFLD, and the prevalence of NAFLD among subjects with BMI > 30 kg/m 2 is four times higher than 461 that among normal-weight subjects (7). Obesity is closely related to insulin resistance. Furthermore, it is associated with 462 hyperinsulinemia and high inflammatory cytokine levels, which can promote the development of hepatocyte steatosis (8,9). On the 463 other hand, NAFLD is considered to be a manifestation of metabolic syndrome, and is an independent risk factor for diabetes and 464 cardiovascular disease (6, 10-12). Therefore, seeking an optimal treatment for NAFLD and related metabolic disorders has become a 465 hot topic for researchers.

466
Lifestyle interventions play a role in improving NAFLD by reducing weight and insulin resistance (13)

482
We propose to conduct a randomized controlled trial to compare the effects of TRE versus DCR on the intrahepatic triglyceride 483 (IHTG) content and metabolic risk factors among obese individuals with NAFLD. We hypothesize that TRE would be more effective 484 than DCR in improving NAFLD and metabolic risk factors.

488
This is a randomized, group-paralleled, observer-masked clinical trial. After screen, all eligible subjects will be randomly 489 assigned to the TRE group or DCR group with an allocation ratio of 1:1 (Figure 1). The duration of intervention included the original 490 designed 6 months and the next 6 months follow-up visits. Participants assigned to the TRE group will be instructed to eat only from 491 8:00 AM to 4:00 PM and only noncaloric beverages will be permitted outside of the eating window during the initial 6-month weight-492 loss phase and the next 6-month follow-up. Participants in the DCR group will be instructed to follow a calorie restriction diet without

538
• Significant alcohol consumption in the past six months (Consumed more than 20 g/day for women or 30 g/day for men);

539
• History of serious cardiovascular or cerebrovascular disease (angina, myocardial infarction or stroke) in the past 6 months;

540
• History of severe gastrointestinal diseases or gastrointestinal surgery in the past 12 months;

542
• Being a smoker or having been a smoker in the 3 months prior to their screening visit;

543
• Taking medications affecting weight or energy intake/energy expenditure in the last 6 months, including weight loss medications,

544
antipsychotic drugs or other medications as determined by the study physician;

545
• Currently participating in weight loss programs or weight change in the past 3 months (> 5% current body weight);

546
• Women who are pregnant or plan to become pregnant;

547
• Patients who cannot be followed for 24 months (due to a health situation or migration);

548
• Patients who are unwilling or unable to give informed consent. assistants who collect study outcome data will be masked to participants' intervention assignment.

555
This trial aims to compare the effect of TRE versus DCR on the improvement of NAFLD and metabolic risk factors. After the 556 initiation of the study, we reviewed our program and prolonged the duration of intervention for 12 months in consideration of purpose 557 of comparisons of long-term effects of TRE versus DCR on NAFLD. Therefore, the whole duration of intervention is 12 months, which 558 included the original designed 6 months and the next 6 months follow-up visits.

560
All participants will be instructed to follow a diet of 1500-1800kcal/d for men and 1200-1500kcal/d for women (40-55% of energy 561 as carbohydrate, 15-20% as protein, 20-30% as fat) (28). Participants assigned to the TRE group will be instructed to eat from 8:00 AM 562 to 4:00 PM and only noncaloric beverages is permitted outside of the eating window. Participants in the DCR group will be instructed 563 to follow a calorie restriction diet without restricting eating period. All participants will be provided with one protein shake (Nutriease,

564
Zhejiang Nutriease Co. Ltd., China) (Table 1) per day for the first 6 months and receive dietary counseling for the duration of the study.

565
All participants will receive written dietary information booklets with portion advice and sample menus and have similar dietary energy 566 restrictions in accordance with Dietary Guidelines for macronutrient intake (28,29). After completing the initial 6 months intervention, 567 participants will be instructed to maintain their diet regimens during the next 6-month follow-up visit.

573
Participants will be encouraged to weigh foods to ensure accuracy of intake. All participants will be required to write their dietary 574 log and record daily food picture and mealtime on a custom mobile study application (App), which will be reviewed daily by study 575 nutritionist. Caloric intake assessment will be conducted by the nutritionist based on each participant's log and daily food photos based 576 on the nutrient content listed in the China Food Composition Table (30). Intervention participants will receive follow-up telephone calls 577 or App message twice per week, and meet with the dietician or nutritionist individually every 2 weeks to assess their adherence to the 578 program and provide suggestions for improvements and personalized energy targets for weight maintenance in the initial 6 months 579 intervention (Table 2).

580
After completing the initial 6 months intervention, participants will be instructed to maintain their diet regimens during the next 6-581 month follow-up visit, and write their dietary log and record food picture and mealtime three times per week. In this phase, participants 582 received follow-up telephone calls or an App message once per week and met with the nutritionist monthly.

587
All participants will be instructed to attend health education sessions monthly. Education sessions content will include weight 588 measurement, dietary recall, dietary instruction (e.g., recipes, sample menus, food list, knowledge about macronutrients counting, 589 nutrition labels reading), behavioral strategies, general health knowledge of NAFLD and metabolic disease (Table 3). All participants 590 will be instructed not to change their physical activity habits throughout the trial.

622
Whole body compositions will be quantified using a whole-body dual x-ray system (Lunar iDXA, GE Healthcare). 623 9.6 Body fat rate:

624
The bioelectrical impedance method will be used to measure body fat rate using the human body fat analyzer (V.Body HBF-371,
Diet counseling and health education X X X X X X X

646
Protection of participants from risks related to the intervention is of paramount concern to the physicians. All participants will be 647 evaluated for their medical history before enrollment. Contact information of the physicians are available for all the participants.

648
All participants should be contacted regularly to evaluate the occurrence, severity, and duration of physical discomfort, including 649 dizziness, fatigue, abdominal pain, headache, appetite change, constipation, or other adverse events during intervention. Evidence of the 650 occurrence of adverse events should be based on participants' self-report rather than suggestive questioning. Any medical adverse events 651 will be recorded and the nature, severity, and cause-effect relationship will be evaluated by physicians and recorded in a timely manner 652 on case report forms.

654
The severity of adverse events is assessed as follows:

655
Mild: usually temporary, and not affecting daily activities;

656
Moderate: causing discomfort and affecting normal activities, thought tolerable and not requiring participants to take any 657 medication.

658
Severe: disrupting daily activities and intolerable, requiring participants to take medication immediately.

659
With regard to the causal-effect relationship, medical adverse events are divided into five categories: definite, probable, possible, 660 unlikely, and unrelated. Only "definite" and "probable" are counted as adverse events.

673
All data will be double-entered by researcher staff. Two sets of databases will be generated and tested for consistency using the 674 SAS program. Whenever inconsistencies are found, the data will be corrected by re-examination of the original case report forms or 675 laboratory reports.

677
Several standardized reports will be generated as follows: 1) participant recruitment and follow-up; 2) demographics; 3) data quality 678 and monitoring; 4) adverse events. These reports will be used for study management. These reports will be blinded to research personnel 679 who collect study outcomes.

682
Data will be analyzed according to participants' randomization assignments, regardless of their subsequent status (intention-to-683 treat). PROC MIXED of SAS statistical software, version 9.4 (SAS Institute Inc), will be used to obtain point estimates and SEs of the 684 treatment effects and to test for differences between treatments. Group differences in the study outcomes will be evaluated using the 685 general linear model for continuous variables and the chi-squared test for categorical variables. A mixed-effects model will be used to 686 assess the effects of diet programs on the change in IHTG content and main outcomes and an autoregressive correlation matrix will be 687 used to correct within-participant correlation for repeated measurements. In this model, participants will be assumed to be random effects,

688
and intervention group, time, and their 2-factor interactions will be assumed to be estimable fixed effects. Multiple imputations for 689 missing data in the multivariable analyses will be conducted using the Markov chain Monte Carlo method. Data will be presented as 690 least-squares means with 95% confidence intervals (CIs). P < 0.05 will be considered statistically significant.

693
All study staff (physicians and nurses) will be trained and certified before the study is initiated. The training program will include 694 instruction on questionnaire administration and anthropometrics (height, weight, waist circumference, and hip girth) and blood pressure 695 measurement. A standard questionnaire will be used, which includes personal information (name, sex, age, place of birth, marital status, 696 occupation, education, household income, etc.), medical history, medication use, family history, lifestyle factors (smoking and drinking), 697 physical activity, dietary habits, and menstrual and reproductive history. Anthropometric measurements and blood pressure will be 698 obtained using a standard protocol. The data, including that for exercise intensity, duration, heart rate, and anthropometric measurements,

699
will be collected and recorded in CRFs by nurses every week. All technologists are experienced and masked to participant randomization.

702
China's Standard for Quality Management of Clinical Trials (GCP) and other relevant laws and regulations on clinical trials will 703 be complied with. The standard quality control process will be carried out at each step of the study, including repeated laboratory 704 measurements in 5-10% of random blind samples, and all data will be entered independently.

705
Establish a research procedure manual (MOP): we will develop a standardized process of data collection, which will include the 706 recruitment of subjects, instructions for the use of tables, intervention programs, index measurement, specimen collection and 707 management, etc., to guide all tabular information entry and diagnosis and treatment processes, and other aspects of the study.

708
Training: all researchers will receive relevant training before the start of the study, including standardized processes, recruitment 709 processes, follow-up, intervention programs, measurement procedures, and the use of MOP. At the same time, regular retraining sessions 710 will be conducted to ensure the quality of research.

711
Quality monitoring and reporting: quality control personnel will regularly review the QC data, including the timeliness of the 712 completion of the visit, data collection, program implementation and data quality.

718
approving the research program, including modification of the research program, monitoring the recruitment and withdrawal process of 719 subjects, implementation of intervention programs, recruitment and intervention of subjects, data collection and quality control, and 720 responding to questions raised by the ethics body review committee (Institutional review board, IRB), etc. The research center will be 721 involved in the recruitment, intervention, data collection and quality control of the subjects. the research center will do randomization 722 and data analysis, and help prepare to report research results and papers for publication. In order to facilitate communication between 723 investigators and researchers, regular working meetings will be held. SC will discuss relevant scientific and management issues in the 724 study with three live meetings a year and monthly conference calls. Among them, important information, such as recruitment progress, 725 data completion and quality, and compliance with intervention programs, will be sent monthly by e-mail to committee members and key 726 staff.

729
This clinical trial must comply with the Helsinki Declaration (1996 edition) and the relevant regulations of Chinese clinical trials.

730
Before the trial begins, the hospital ethics committee of the research unit shall review the trial plan and issue the approval document 731 before the implementation of the trial plan.

732
For all research program amendments (excluding administrative amendments and amendments that have no impact on the 733 implementation of subjects, data or trials), the amendment and the applicable informed consent must be submitted to the Ethics

734
Committee for approval immediately prior to the implementation of these changes. The researcher is responsible for ensuring that the 735 conditions approved by the study are met and that the trial correction program or serious adverse events are reported to the ethics 736 committee or the corresponding organization as required by the ethics committee.

737
All subjects will be given the opportunity to read the entire informed consent document and, by the researcher or authorized 738 researcher, to provide the subjects, in writing, with a complete and comprehensive presentation of the background, purpose, research 739 methods, research process, test items to be involved, individual rights and obligations, possible risks, and possible benefits of the research 740 process. The subjects had enough time to ask questions, and the researchers needed to provide truthful and accurate answers in plain 741 language. The subjects had sufficient time to discuss with other family members whether to participate in the study. The subjects should 742 know that they can withdraw from the study at any time without any reason; the subjects agree to collect and apply the data related to 743 the study and are willing to cooperate with the follow-up. When they exit, the subjects are asked whether they agree to apply the collected 744 data. Participants will be required to sign informed consent before they can be selected for clinical trials. Informed consent shall be kept 745 for reference as one of the original materials for clinical trials.

748
The clinical study is expected to take about 3 years after registration in the clinicaltrials.gov (Identifier: NCT03786523 and 749 NCT04988230), the trial is expected to take about 3 years to carry out this clinical study, in which MOP development and personnel 750 training and subject recruitment time is 1 year. Intervention and data collection cycle is 18 months, data entry and analysis and paper 751 writing lasted about 0.5 years.